Milk Micelles Research Articles

Correction to: Microstructural observation of casein micelles in milk by cryo-electron microscopy of vitreous sections (CEMOVIS)

Correction to: Microstructural observation of casein micelles in milk by cryo-electron microscopy of vitreous sections (CEMOVIS)

Enzymatic hydrolysis and extensive heat treatment induce distinct modifications of cow's and camel milk proteins of relevance for production of infant formula

Camel milk is an alternative to cow's milk as protein source for infant formula. Reduced allergenicity is usually obtained through enzymatic hydrolysis of milk proteins. Heat treatment is another route to modify allergenicity. This study investigated the effect of enzyme hydrolysis and extensive heat treatment on the size, aggregation mechanisms, and modifications of camel milk proteins in comparison to the effect on cow's milk proteins. Disulphide bonds stabilized casein micelles in cow's milk but not in camel milk. The formation of large stable complexes by extensive heat treatment was prominent in cow's milk and not in camel milk. In both milks heat treatment led to equal levels of Maillard reaction, whereas sugar independent cross-linking was less pronounced in camel milk. Low sequence identity, different relative composition, and complete absence of β-lactoglobulin in camel milk are likely explanations for the different properties of cow's and camel milk. Production of infant formula based on camel milk should accordingly employ process parameters specifically adapted to this type of milk.

Effect of temperature and protein concentration on the protein types within the ultracentrifugation supernatant of liquid micellar casein concentrate

Liquid micellar casein concentrate (MCC) is an ideal milk-based protein ingredient for neutral-pH ready-to-drink beverages. The texture and mouthfeel of liquid MCC-based beverages depend on the beverage protein content, as well as the composition of soluble proteins in the aqueous phase around the casein micelle. The objective of this study was to determine the composition of soluble proteins in the aqueous phase around the casein micelles in skim milk and liquid MCC containing 7.0% and 11.6% protein content. Skim milk was pasteurized and concentrated to 7% protein content by microfiltration and then to 18% protein content by ultrafiltration. The 18% MCC was then serially diluted with distilled water to produce 11.6% and 7.0% protein MCC. Skim milk, 7.0% MCC, and 11.6% MCC representing starting materials with different protein concentrations were each ultracentrifuged at 100,605 × g for 2 h. The ultracentrifugation for each of the starting materials was performed at 3 different temperatures: 4°C, 20°C, and 37°C. The ultracentrifugation supernatants were collected to represent the aqueous phase around the casein micelle in MCC solutions. The supernatants were analyzed by Kjeldahl to determine the crude protein, casein, and casein as a percentage of crude protein content, and by sodium dodecyl sulfate PAGE to determine the composition of the individual proteins. Most of the proteins in MCC supernatant (about 45%) were casein proteolysis products. The remaining proteins in the MCC supernatant consisted of a combination of intact αS-, β-, and κ-caseins (about 40%) and serum proteins (14-18%). Concentrations of αS-casein and β-casein in the supernatant increased with decreasing temperature, especially at higher protein concentrations. Temperature and interaction between temperature and protein explained about 80% of the variation in concentration of supernatant αS- and β-caseins. Concentration of supernatant κ-casein, casein proteolysis products, and serum protein increased with increasing MCC protein concentration, and MCC protein concentration explained most of the variation in supernatant κ-casein, casein proteolysis products, and serum protein concentrations. Predicted MCC apparent viscosity was positively associated with the dissociation of αS- and β-caseins. Optimal beverage viscosity could be achieved by controlling the dissociation of these proteins in MCC.

Effect of green tea extract on heat-induced protein interactions and rennet coagulation in milk

The impact of green tea extract (GTE) on heat-induced milk protein interactions and enzymatic coagulation of milk was investigated. GTE addition affected the renneting process, more apparent for heat-treated samples. The enzymatic phase of rennet coagulation was negatively affected by GTE, with a significantly lower amount of κ-casein hydrolysis for intense heat-treated samples. Gel characteristics of raw milk were unaffected by GTE, but mild heat-treated samples with GTE displayed decreased gel firmness and increased water holding capacity compared with control samples. Model studies conducted with purified β-lactoglobulin A and κ-casein revealed that GTE caused a significant impact on interactions between denatured β-lactoglobulin A and κ-casein. Heat-induced large aggregate/complex formation between β-lactoglobulin A and κ-casein in milk samples was not significantly affected, highlighting the importance of the reaction medium. The presence of casein micelles in milk, which are strong candidates for phenolic binding, may ultimately impact protein–protein interactions.

Transmembrane Pressure during Micro- and Diafiltration of Milk Affects the Release of Non-Sedimentable Caseins.

Membrane filtration, especially in combination with diafiltration, can affect the colloidal structure of casein micelles in milk and concentrated milks. The partial dissociation of casein proteins from the casein micelles into the serum phase has been shown to depend on diafiltration conditions. This dissociation can affect the technological functionality of the milk concentrates. The present study aimed at determining the contribution of the gel layer deposited onto the membrane during filtration in the colloidal equilibrium between soluble and micellar caseins. Skimmed milk was concentrated by microfiltration combined with diafiltration using a cross-flow spiral-wound membrane at two transmembrane pressure (TMP) levels, causing differences in the extent of the gel layer formed. Non-sedimentable casein aggregates were formed to a greater extent at a low TMP compared to a high operating TMP. This difference was attributed to the greater compression of the deposit layer during filtration at a high TMP. This study contributes new knowledge to the understanding of how to modulate the functionality of milk concentrates through the control of processing conditions.

Developmental changes in proteins of casein micelles in goat milk using data-independent acquisition-based proteomics methods during the lactation cycle.

Casein micelles (CM) play an important role in milk secretion, stability, and processing. The composition and content of milk proteins are affected by physiological factors, which have been widely investigated. However, the variation in CM proteins in goat milk throughout the lactation cycle has yet to be fully clarified. In the current study, milk samples were collected at d 1, 3, 30, 90, 150, and 240 of lactation from 15 dairy goats. The size of CM was determined using laser light scattering, and CM proteins were separated, digested, and identified using data-independent acquisition (DIA) and data-dependent acquisition (DDA)-based proteomics approaches. According to clustering and principal component analysis, protein profiles identified using DIA were similar to those identified using the DDA approach. Significant differences in the abundance of 115 proteins during the lactation cycle were identified using the DIA approach. Developmental changes in the CM proteome corresponding to lactation stages were revealed: levels of lecithin cholesterol acyltransferase, folate receptor α, and prominin 2 increased from 1 to 240 d, whereas levels of growth/differentiation factor 8, peptidoglycan-recognition protein, and 45 kDa calcium-binding protein decreased in the same period. In addition, lipoprotein lipase, glycoprotein IIIb, and α-lactalbumin levels increased from 1 to 90 d and then decreased to 240 d, which is consistent with the change in CM size. Protein-protein interaction analysis showed that fibronectin, albumin, and apolipoprotein E interacted more with other proteins at the central node. These findings indicate that changes in the CM proteome during lactation could be related to requirements of newborn development, as well as mammary gland development, and may thus contribute to elucidating the physical and chemical properties of CM.

Kinetics of heat-induced interactions among whey proteins and casein micelles in sheep skim milk and aggregation of the casein micelles

The interactions among the proteins in sheep skim milk (SSM) during heat treatments (67.5-90°C for 0.5-30 min) were characterized by the kinetics of the denaturation of the whey proteins and of the association of the denatured whey proteins with casein micelles, and changes in the size and structure of casein micelles. The relationship between the size of the casein micelles and the association of whey proteins with the casein micelles is discussed. The level of denaturation and association with the casein micelles for β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) increased with increasing heating temperature and time; the rates of denaturation and association with the casein micelles were markedly higher for β-LG than for α-LA in the temperature range 80 to 90°C; the Arrhenius critical temperature was 80°C for the denaturation of both β-LG and α-LA. The casein micelle size increased by 7 to 120 nm, depending on the heating temperature and the holding time. For instance, the micelle size (about 293 nm) of SSM heated at 90°C for 30 min increased by about 70% compared with that (about 174.6 nm) of unheated SSM. The casein micelle size increased slowly by a maximum of about 65 nm until the level of association of the denatured whey proteins with casein micelles reached 95%, and then increased markedly by a maximum of about 120 nm when the association level was greater than about 95%. The marked increases in casein micelle size in heated SSM were due to aggregation of the casein micelles. Aggregation of the casein micelles and association of whey protein with the micelles occurred simultaneously in SSM during heating.

Natural Association of Lysozyme and Casein Micelles in Human Milk.

Using reversed phase high-performance liquid chromatography with ultraviolet (UV) detection and electrospray ionization (ESI)-quadrupole time-of-flight mass spectrometry (RP-HPLC-UV-ESI-Q-TOF), the lysozyme content in the milk of 10 volunteering mothers was quantified, ranging from 29 to 96 μg/mL. Following ultracentifugation, it was found that the lysozyme in human milk, unlike other whey proteins, is mainly bound to casein micelles (ca. 75%). The enzymatic activity of human lysozyme, measured as lytic activity against cell walls of Micrococcus lysodeikticus, was similar for the micelle-bound and free protein, indicating that the micellar structure should not affect the antibacterial activity of lysozyme. The results indicate that lysozyme is an integral component of casein micelles in human milk.

Effect of alkalinization and ultra-high-pressure homogenization on casein micelles in raw and pasteurized skim milk

Mechanical and physicochemical treatments of milk induce structural modifications of the casein (CN) micelles, affecting their techno-functional properties in dairy processing. Here, we studied the effect of alkalinization and ultra-high-pressure homogenization (UHPH) on CN micelles in raw skim milk (rSM) and pasteurized skim milk (pSM). The pH of both skim milks (approximately 6.7) was adjusted to 8.5 and 10.5 before UHPH at 100, 200, and 300 MPa. The structural changes of the CN micelles during the treatments were assessed using laser diffraction, transmission electron microscopy, and turbidity measurements. Finally, ultracentrifugation (70,000 × g for 1 h at 20°C) was carried out to evaluate the protein's distribution between the supernatant (serum phase) and the pellet (colloidal phase) by gel electrophoresis and protein concentration measurement. Alkalinization of both skim milks induced a significant reduction in turbidity, whereas an increase of the average particle size was observed, the effect being more severe in pSM than rSM. At alkaline pH, more proteins were recovered in the serum phase, which suggested that the CN underwent major rearrangements into nonsedimentable CN forms of various sizes, as confirmed by transmission electron microscopy. The amount of CN found in the serum phase at pH 8.5 also increased with the UHPH pressure. Although UHPH did not influence the average CN micelle size at pH 6.7 and 8.5, a pressure-dependent decrease was observed at pH 10.5 for both skim milks. The structural changes of the CN micelles observed in this study throughout the combination of alkalinization and UHPH could be of interest for developing new dairy ingredients with improved functionality.

Kinetics of pepsin-induced hydrolysis and the coagulation of milk proteins

Hydrolysis-induced coagulation of casein micelles by pepsin occurs during the digestion of milk. In this study, the effect of pH (6.7-5.3) and pepsin concentration (0.110-2.75 U/mL) on the hydrolysis of κ-casein and the coagulation of the casein micelles in bovine skim milk was investigated at 37°C using reverse-phase HPLC, oscillatory rheology, and confocal laser scanning microscopy. The hydrolysis of κ-casein followed a combined kinetic model of first-order hydrolysis and putative pepsin denaturation. The hydrolysis rate increased with increasing pepsin concentration at a given pH, was pH dependent, and reached a maximum at pH ∼6.0. Both the increase in pepsin concentration and decrease in pH resulted in a shorter coagulation time. The extent of κ-casein hydrolysis required for coagulation was independent of the pepsin concentration at a given pH and, because of the lower electrostatic repulsion between para-casein micelles at lower pH, decreased markedly from ∼73% to ∼33% when pH decreased from 6.3 to 5.3. In addition, the rheological properties and the microstructures of the coagulum were markedly affected by the pH and the pepsin concentration. The knowledge obtained from this study provides further understanding on the mechanism of milk coagulation, occurring at the initial stage of transiting into gastric conditions with high pH and low pepsin concentration.

A study on the structure of bovine casein micelles in milk using quantum beams

A study on the structure of bovine casein micelles in milk using quantum beams

Chemical interactions among caseins during rennet coagulation of milk

Rennet milk curds were prepared under 4 different temperature and acidity conditions. The development of different types of inter-protein chemical bonds (disulfide, hydrophobic, electrostatic, hydrogen, and calcium bridges) was monitored for 60 min after curd cutting. Hydrophobic inter-protein interactions originally present in casein micelles in milk were substituted by electrostatic, hydrogen, and calcium bonds throughout the curd curing period. Disulfide bonds were not disturbed by the experimental conditions employed in the study, remaining at a constant level in all studied treatments. Acidification of curds increased the availability of soluble ionic calcium, increasing the relative proportion of calcium bridges at the expense of electrostatic-hydrogen bonds. Although pH defined the nature of the interactions established among proteins in curd, temperature modified the rate at which such bonds were formed.

Influence of Chymosin on Physicochemical and Hydrolysis Characteristics of Casein Micelles and Individual Caseins.

The effects of chymosin on the physicochemical and hydrolysis characteristics of casein micelles and individual caseins were investigated. Adding 0.03 units of chymosin/mL led to the casein micelles in skim milk coagulating after a 3 h incubation period at 30 °C. SDS–PAGE investigation showed that β-CN, κ-CN, αs-CN, and a portion of β-lactoglobulin (β-LG) in the milk supernatant fraction (MSF) were precipitated into the milk pellet fraction (MPF). The mean particle size of the MSF with chymosin decreased from 254.4 nm to 179.2 nm after a 3 h incubation period. Mass spectrometry and SDS–PAGE analysis suggested that chymosin hydrolyzed individual β-CN, κ-CN, and αs-CN, but not β-LG. Chymosin hydrolysis led to a decrease in the molecular weights of the hydrolyzed β-CN, κ-CN, and αs-CN. Particle size analysis indicated that there was no difference in the particle size distribution of hydrolyzed β-CN and αs-CN. Moreover, our outcomes demonstrated that the hydrolysis of κ-CN by chymosin occurs before that of β-CN and αs-CN.

Sodium caseinate hinders chymosin-induced aggregation of caseins in concentrated milk: The role of soluble caseins and calcium ions

Concentrated casein micelle suspensions show an altered balance between the colloidal and soluble phases compared with native skim milk. The objective of this research was to probe the role of such a change on the chymosin-driven destabilization of casein micelles. Skim milk was gently concentrated by osmotic stressing to increase the volume fraction of the micelles while maintaining a constant ionic composition. In situ turbidity measurements demonstrated that the secondary stage of gelation was hindered in the concentrated suspensions. Addition of ionic calcium overcame this inhibition, whereas restoring the original concentration by redilution did not. This work clearly demonstrated that calcium plays a major role in decreasing electrostatic repulsion in casein micelles, but also showed the importance of noncolloidal proteins in altering the gelling functionality of casein micelles in concentrated milk. Additional calcium induces aggregation of these soluble caseins, restoring gelation in concentrates.

Interplay of interactions between micelles and fibrils of casein proteins

Kappa-casein (κCΝ) and beta-casein (βCN) are two of the four milk proteins that constitute the milk micelles (casein micelles), ~250 nm aggregates held by calcium-phosphate bridges, designed by nature to deliver nutrients to the neonate. κCN is localized at the surface of the micelle, while βCN, together with other caseins, is located inside the micelle. At pathological conditions κCN undergoes fibrilization. In an earlier study we showed that fibrils formation and growth can be hindered in mixed κCN/βCN solutions by the formation of classical core-shell mixed micelles. The present study focuses on a later stage, where κCN fibrils are already formed. Using ITC, we find strong adsorption of βCN onto the fibrils, probably in the form of micelles as indicated by Cryo-TEM and SAXS. The results of the interaction between κCN fibrils and another amphiphile, Lutrol (Pluronic F-127), also demonstrated the adsorption. Some differences were found in the manner of adsorption of the amphiphiles. βCN adsorption is manifested at relatively high concentrations, after its demicellization/micellization. Lutrol, on the other hand, adsorbs already upon initial ITC injections, in parallel with demicellization, as indicated by splitting of the peaks. By deconvolution of the splitting peaks, the contributions of demicellization and adsorption of Lutrol were resolved. We conclude that the polymer adsorption is an additional factor suppressing κCN fibrillization and fibril growth. The decorated κCN fibrils may be considered as a new functional material.

The relationship between ultra-small-angle X-ray scattering and viscosity measurements of casein micelles in skim milk concentrates

Skim milk concentrates have important applications in the dairy industry, often as intermediate ingredients. Concentration of skim milk by reverse osmosis membrane filtration induces water removal, which reduces the free volume between the colloidal components, in particular the casein micelles. Thermal treatment before or after concentration impacts the morphology of casein micelles. These changes affect the flow behavior and viscosity, but the consequences for supermicellar structure have not been elucidated. In the present study, skim milk concentrates with different total solid contents from 8.7% (control) up to 22.8% (w/w), prepared by reverse osmosis membrane filtration of non-heated and pasteurized skim milk, were heat treated at 75 °C for 18 s, and compared with non-heated concentrates. The structure of the concentrates was studied using Ultra Small Angle X-ray Scattering (USAXS), and the viscosity of concentrates was measured. The USAXS intensity I(q) was fitted at small and intermediate q-regions (0.0005 < q < 0.003 Å−1 and 0.0035 < q < 0.03 Å−1, respectively) with a power law. The value of the power law exponent was used to assess the heat- and concentration-induced aggregation of the milk solids and correlate it with the apparent viscosity. The results showed that increased viscosity of skim milk concentrates, due to water removal and heat-load, can be explained by increased aggregation of the casein micelles into elongated aggregates and increased smoothening of the casein micelle surface.

Оценка влияния полиморфизма гена κ-казеина в сухом молоке на технологические свойства кислотно-индуцированных молочных гелей

Introduction. Yoghurt has become one of the most popular acid-induced dairy products in the world. Consumers see yoghurt not only as a tasty, protein-rich, and calcium-fortified dessert, but also as a product that improves intestinal microflora and prevents obesity, metabolic syndrome, type II diabetes, and cardiovascular diseases. The stability of the structure and consistency of yoghurt directly depend on the composition and properties of raw materials, including genetic and technological factors and compliance with temperature storage conditions. Yoghurt formulations include various dairy raw materials, e.g. milk powder. The research objective was to assess the effect of κ-casein gene polymorphism in milk powder on the technological properties of acid-induced milk gels when simulating different temperature storage conditions. Study objects and methods. The research featured yoghurt samples prepared from milk powder of CSN3 gene (AA and BB). Model systems of yoghurt were prepared from dry bulk milk, mixed by mass fraction of protein in the ratio of AA2:BB2 as 75:25, 50:50, and 25:75%, respectively. The experiment involved standard methods, optical methods, dynamic viscometry, and PCR-RFLP. Results and discussion. As the mass fraction of BB dry bulk milk increased, the structural and mechanical properties, dimensional stability, and surface tension increased, too. As the storage temperature fell from 4 ± 2 to 12 ± 2°C, the structural and mechanical properties, dimensional stability, surface tension, and moisture-holding ability decreased while maintaining the previously established dependencies. The CSN3 gene polymorphism proved to have no effect on the curd tension after fermentation. Significant differences between the allelic variants AA and BB became obvious only after complete cooling and structuring of the product. Conclusion. The obtained experimental results and the analysis of related publications suggested an indirect effect of the κ-casein gene polymorphism on the structural and mechanical properties, associated with a genetic effect on the average diameter of casein micelles in the original milk and the resulting biochemical and isothermal processes. The research made it possible to assess the effect of the CSN3 gene polymorphism on the technological properties of dry milk during its processing into fermented milk products.

A novel approach for characterisation of stabilising bonds in milk protein deposit layers on microfiltration membranes

The gel formation mechanism of low-pressure casein gels during the filtration of skim milk remains unknown this far. We investigated the prevailing bond types in this gel and correlated these findings with deposit layer properties and filtration performance. At high pH and low transmembrane pressure, calcium bridges predominated among the bond types stabilising the deposit layer gel. However, there were less calcium bridges present than within or between casein micelles in skim milk. With a decreasing amount of calcium bridges at low pH and higher transmembrane pressures, the deposit layer turned into a dense and mushy gel, which impaired solvent and solute permeability. Additionally, ionic and hydrogen bonds, as well as disulphide bonds, were formed between the highly concentrated deposited proteins during gelation. Hydrophobic interactions played a negligible role. We concluded that intramicellar calcium bridges are associated with a voluminous micelle and a stable gel backbone, which benefits filtration efficiency.

An alternative analysis of contrast-variation neutron scattering data of casein micelles in semi-deuterated milk.

Contrast-variation small-angle neutron scattering (CV-SANS) is an excellent way to determine the structure of complex, hierarchical colloids, including self-assembled biological systems. In these experiments, the scattering length density of solvents is changed (by varying the ratio of water or [Formula: see text] and heavy water or [Formula: see text]) to highlight or mask scattering from different components in the system. This approach has been used with synthetic colloids, but it is also increasingly being used in the biological and food sciences. Perhaps the most studied food colloid is the "casein micelle," a self-assembled nanometer-scale colloid of the structure-forming casein protein in milk. CV-SANS data available in the literature are typically analyzed using approximations, which may be invalid for casein micelles, as they have been shown to be sticky spheres. To assess the applicability of this approximate approach, a comprehensive set of CV-SANS data from casein micelles in diluted milk was reanalyzed using a model-based approach, where the casein micelles were formally treated as interacting spheres. In general, the conclusions of the previous study are reproduced, but this new approach makes it more straightforward to distinguish the different components in milk and can be applied to any dairy sample with known form of interparticle interactions, which offers the possibility of studying semi-deuterated milk at its native concentration.

Comparative study on the microstructure and functional properties of casein in goat milk processed by different methods

SummaryThis work compared the thermal stability, microstructure and functional properties of casein (CN) in goat milk processed by different methods, including homogenisation, low‐temperature long‐time (LTLT), high‐temperature short‐time (HTST), ultrapasteurisation (UP) and ultrahigh temperature (UHT) treatment. The greatest development of CN‐whey protein complexes was observed in LTLT goat milk and the least in HTST goat milk. The CN micelles in LTLT, UP and UHT milk were more obviously changed than the CN micelles in HTST milk, and the random coil content increased significantly after the homogenisation, UP and UHT treatments. The homogenisation had a significant effect on the functional properties of CN. In addition, the most obvious changes in the properties of CN were observed in LTLT and UHT goat milk. The results suggested that homogenisation, pasteurisation and UHT can cause effective changes in the functional properties of CN in goat milk that facilitate its application in the milk industry.